Mitochondria-targeted half-sandwich iridium(iii)-Cp*-arylimidazophenanthroline complexes as antiproliferative and bioimaging agents against triple negative breast cancer cells MDA-MB-468

To reduce the side effects of marketed cancer drugs against triple negative breast cancer cells we have reported mitochondria targeting half-sandwich iridium(iii)-Cp*-arylimidazophenanthroline complexes for MDA-MB-468 cell therapy and diagnosis. Out of five Ir(iii) complexes (IrL1–IrL5), [iridium(iii)-Cp*-2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline]PF6 (IrL1) has exhibited the best cytoselectivity against MDA-MB-468 cells compared to normal HaCaT cells along with excellent binding efficacy with DNA as well as serum albumin. The subcellular localization study of the complex revealed the localization of the compound in cytoplasm thereby pointing to a possible mitochondrial localization and consequent mitochondrial dysfunction via MMP alteration and ROS generation. Moreover, the IrL1 complex facilitated a substantial G1 phase cell-cycle arrest of MDA-MB-468 cells at the highest tested concentration of 5 μM. The study verdicts support the prospective therapeutic potential of the IrL1 complex in the treatment and eradication of triple negative breast cancer cells. These results validate that these types of scaffolds will be fairly able to exert great potential for tumor diagnosis as well as therapy in the near future.


Introduction
Nowadays, breast cancer prevails over most of the deadly life threats towards women from all over the world. 11.7% of new cases of cancer diagnosed in 2020 were female breast cancer which is the highest number of cases among all types of cancer reported. 1 This disease is heterogenous at the molecular level but, as a consequence of diligent research over past decades, chances of healing have been increased by 70-80% in breast cancer patients when the cancer is non-metastatic and treated in the early stage. Over the years of research a few categorisations of tumours were rened based on the alterations at the molecular level. 2 Current clinical practices involve an alternate classication of 5 types that includes triple negative breast cancer (TNBC) without expression of ER, PR or HER2. TNBC is adenoid cystic and metaplastic in nature with a poor prognosis prole. 2 A thorough investigation of medical literature reveals that the term, "triple negative breast cancer", was rst mentioned in 2005 by Brenton and coworkers. 3 However, in the present situation 12-17% of all reported breast cancer cases have been recognised under this class.
It has been challenging to create an effective treatment regime for TNBC patients as this aggressive subtype does not respond to the HER2 targeting drugs or hormonal therapy. Consequently, chemotherapy remains as the main systemic treatment option even though TNBC develops resistance easily to existing targeted medicines like trastuzumab. Potential molecular targets for TNBC may include EGFR (a surface receptor), PARP1 (poly ADP-ribose polymerase 1) and DNA. The phenotypic similarity of TNBC to BRCA-1 associated malignancy helped researchers to develop few targeted cytotoxic agents currently which may lead to a new horizon of TNBC therapeutics. In 2020 FDA approved trodelvy (sacituzumab govitecanhziy) which is a topoisomerase inhibitor conjugate antibody directed to Trop-2 receptor for metastatic TNBC and for the rst-time improved progression of overall survival was witnessed. Triple negative breast cancer caused by BRCA1 and BRCA2 mutation can be a potential target of DNA damaging chemotherapeutics and a number of clinical data leads to the suggestion that involving platinum-based chemotherapeutics may be use in standard treatment regime of early stage as well as advanced TNBC. 4 As reported in earlier scientic research, platinum salts like cisplatin, carboplatin or oxaliplatin are able to initiate a platinum-DNA adduct formation followed by DNA damage in cancer cells leading towards apoptosis of the targeted cancer cells. BRCA1 decient breast cancer showed sensitivity towards platinum and gemcitabine neoadjuvant treatment but with poor chance of progression free survival. 5 A clinical study, reported by von Minckwitz in 2013 involving 315 early stage TNBC patients, let out the fact that a neoadjuvant treatment regime comprising doxorubicin and carboplatin achieved 59% pathologic complete response rate. 6 However, a combination therapy of gemcitabine/carboplatin given to advanced metastatic TNBC patients in a trial scored 34% objective response rate and progression free survival of 5.1 months. 7 Although cisplatin and its analogical platinum complexes showed slight prospect in TNBC treatment they have exerted some major drawbacks like toxicity, chemoresistance and narrow activity window. 8 This fact motivated broader area of investigation on efficacy of other metal complexes to target metastasis and defective cell proliferation. A handful of gold, iron, copper, ruthenium, rhodium and iridium complexes happened to be synthesized and tasted against breast cancer cell lines in vitro or in vivo and found to manifest excellent outcomes. 9 Of late, Ir(III) based metal complexes have been fascinating the mind of the investigators for being reconnoitered as highly active anticancer agents with their excellent photoluminescence property possessing high photostabilities, long-lived excited triplet states aer quick singlet to triplet intersystem crossing (ISC), larger Stokes' shi associated with high quantum yields. 10,11 Furthermore, the higher oxidation state of iridium metal, ability to display a wide range of ligand substitution, considerable redox properties, exible structural features and stability towards cancer cell environments has enabled them to be used in preparing anticancer metal complexes. 10,11 Instead, bioactivities of metal complexes are also reliant on the structure of the ligands. Arylimidazophenthroline compounds are well known as pertinent probes of DNA structure having the capability of disentangling the double stranded DNA through strong intercalative interaction. 12 Encouraged by our previous work with ruthenium and arylimidazophenthroline herein we have intended to design ve iridium(III)-Cp* N^N metal complexes with planar and p-extended arylimidazophenanthroline moiety as ligand having strong metal binding capacity (Fig. 1). [13][14][15][16][17][18] and evaluated their antiproliferative activity against MDA-MB-468 TNBC cells that are EGFR +ve, TGF alpha +ve, high in Ki67. These complexes exhibited dual properties like (i) killing of cancer cells via DNA damage (ii) mitochondrial dysfunction by ROS production. 19 The intrinsic phosphorescence property of these Ir(III) complexes are also helpful for cellular imaging and tracking of drug accumulation in subcellular organelles. 20

Results and discussion
Chemistry Synthesis and characterization. A series of imidazo [4,5-f] [1,10]phenanthroline ligands (L1-L5) was prepared by treating an equimolar mixture of 1,10-phenanthroline-5,6-dione and different aromatic carboxaldehydes (1)(2)(3)(4)(5) in the presence of ammonium acetate and glacial acetic acid, following the same procedure as mentioned in our previous communication. 21 Further to prepare Ir(III)-Cp*-imidazophenanthroline complexes (IrL1-IrL5), [(C 5 (CH 3 ) 5 IrCl 2 )] 2 was added to the prepared ligands (L1-L5) in a 1 : 2 ratio in methanol and stirred at room temperature for 2 h. Aer a change in colour from light yellow to orange, 2.5 equivalents of NH 4 PF 6 were added and stirred again for 90 min (Scheme 1). The complexes [Cp*Ir(L1-L5)Cl]PF 6 labelled as IrL1-IrL5 were obtained in good yield (92-95%). The structures of all the complexes (IrL1-IrL5) were analysed via 1 H, 13 C, 19 F and 31 P NMR, and mass spectroscopy. The complex IrL1 displayed a characteristic singlet peak at 1.74 ppm, corresponding to the ve methyl groups of pentamethylcyclopentadiene. The protons of complex IrL1 experienced a considerable downeld effect upon attachment to the iridium Cp* precursor. In the 13 C NMR spectrum, the ligand carbons appeared at around d 125.8-153.2 ppm. The aliphatic CH 3 carbons peaks were observed at d 8.7 ppm and aromatic CH carbons peaks were observed at d 89.6 ppm. In the 19 F NMR  Electronic absorption (UV-visible) and uorescence study. The absorption and emission spectra of all the complexes (IrL1-IrL5) at 298 K were recorded in a DMSO-water (1 : 1) solvent system, as shown in Fig. 2. The photophysical data is summarized in Table 1. The characteristic intraligand (p-p*) transitions (N^N ligands) appeared at 250-350 nm and metal to ligand charge transfer ( 1 MLCT) at 360-400 nm. 22,23 Among the complexes, we observed the maximum absorption in the 1 MLCT region for the benzothiazole derivative (IrL5). In the emission spectra, we observed the MLCT emission of all the complexes in the range of 350-525 nm (Fig. 2). Similar to the absorption spectra, the emission for the anthracene derivative is the most intense because of its strong p conjugation. Using the emission spectral data, the quantum yield of these complexes was calculated. These complexes didn't show remarkable quantum yield, complex IrL1 showed moderate quantum yield (0.002) though for the MLCT transition (Table 1).
Solubility, lipophilicity and conductivity study. Both hydrophilicity and lipophilicity studies were performed to determine the tumour-inhibiting potential of the metal complexes. These complexes were highly soluble in DMSO and moderately soluble in H 2 O, MeOH, EtOH and CH 3 CN. Furthermore, they were soluble in the range of 0.6-0.8 mg per mL of 10% DMSO in DMEM, 10 : 90 v/v (comparable to cell media) at 25 C ( Table 1). The lipophilicity of these complexes was determined by performing an n-octanol/water partition coefficient (log P o/w , where P o/w ¼ the octanol/water partition coefficient) study using the shake ask method (Table 1). 24 The experimental log P o/w values of these complexes were determined to be in the range of 0.27-0.34 (Table 1). Complex IrL2 exhibited the highest log P o/w due to the hydrophobic nature of its anthracene group. The lowest log P o/w value was observed for compound IrL3 because it's hydrophilic chromone group. The iridium complexes IrL1-IrL5 exhibited molar conductance values in the range of $7-9 S m 2 M À1 in pure DMSO. Furthermore, their molar conductance increased in 10% DMSO ($22-36 S m 2 M À1 , Table 1), suggesting Scheme 1 Synthesis of (h 5 -Cp*)iridium(III)-imidazophenanthroline complexes.  Stability study of the complexes by UV-vis spectroscopy. The stability studies of complex IrL1 were conducted in six different solvents, i.e. aqueous DMSO (H 2 O : DMSO ¼ 9 : 1) and aqueous GSH medium (Fig. 3) respectively in presence or absence of different concentrations of NaCl. It is essential that the complexes remain stable in the biological environments of cells, and thus the stability studies were performed.
The obvious change in absorbance ($15-30% decrease aer 24 h) with time in aqueous DMSO clearly revealed the moderate dissociation of the -Cl ligand from the Ir(III) complexes followed by aqua complex formation, which was also quantitatively determined based on the observed molar conductivity of the complexes (IrL1-IrL5) in aqueous DMSO and it favours DNA covalent binding as well. It has been reported that many cancer cells become resistant to various drugs by increasing their cellular glutathione level. 28 Hence, to determine the effect of GSH on the reported complexes, a stability study was performed in the presence of excess (10 eq.) glutathione (GSH) via time-dependent UV spectroscopy. Table 1 Photophysical characterization, solubility, lipophilicity and conductivity study of the complexes (IrL1-IrL5)  However, GSH didn't have much impact on the stability of metal complex and hence resistivity may not be induced against this complex in cancer cells containing high level of GSH. Cyclic voltammetry. The CV response of ten-continuous cycle shows a well-dened reversible redox peak at E 0 ¼ À0.485 AE 0.005 V versus Ag/AgCl with a peak separation potential of DE p value is 0.121 AE 0.005 (DE p ¼ E pa À E pc ). Peak current (i pa and i pc ) of the redox peak was found to increasing linearly which ensures the high stability and co-ordination of the metal-ligand complex (Fig. 4).

Biology
In vitro cytotoxicity study. The in vitro cytotoxicity of complexes IrL1-IrL5 and cisplatin were investigated using the typical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol against a panel of triple negative cancer cell line MDA-MB-468. The cells were initially incubated with the all test compounds at concentrations ranging from 0.5-50 mM for 48 h in vitro.
All the complexes exhibited signicant cytotoxicity with IC 50 values of 3.673 mM, 20.35 mM, 20.27 mM, 5.202 mM, 4.412 mM for IrL1, IrL2, IrL3, IrL4 and IrL5 respectively (Fig. 4). The dose dependent cytotoxic effects of these complexes were evident on MDA-MB-468 with IrL1 exhibiting the most potent effects thereby exhibiting the lowest IC 50 value of 3.67 mM (Fig. 5). Consequently, the IrL1 complex doses of 1 mM, 3 mM and 5 mM were selected for further experimental analysis. Furthermore, the possible cytotoxic effects of IrL1 on normal cell line were also examined on the immortalized human keratinocyte cell line HaCaT. The IC 50 value of IrL1 on HaCaT   is found to be 11.42 mM, which is signicantly $3 times higher than the IC 50 value of the complex on the breast cancer cell line MDA-MB-468, suggesting the low cytotoxic effects of the same on normal cells and safety implications to use the compound for selective therapeutics at lower concentrations (Fig. 4).

DNA binding studies
UV absorption method. To design effective chemotherapeutic drugs, it is essential to explore the interactions of metal complexes with DNA. Complex IrL1 displayed strong absorption band at 297 nm. DNA base pairs such as purine (adenine and guanine) and pyrimidine (cytosine and thymine) analogues are responsible for electronic transitions. Upon the addition of CT-DNA in increasing concentration from 5 mM to 60 mM, we observed a hyperchromic shi in p-p* region and hypochromic shi at the MLCT region resulting in the appearance of an isosbestic point, which indicates the prevalence of covalent interaction of the complex with DNA together with the intercalative mode of interaction (Fig. 6).
EtBr quenching study. The competitive binding of compounds IrL1 to CT-DNA was studied via uorescence spectroscopy using ethidium bromide (EtBr) as the uorophore. We clearly observed a gradual decrease in the uorescence intensity of the EtBr bound DNA in the presence of the complexes since they displaced EtBr from DNA, and consequently got bound between the base pairs of the DNA, suggesting the intercalative binding mode of action, as observed in Fig. 7. Intrinsic binding constant (K b ), Stern-Volmer quenching constant (K sv ) and apparent binding constant (K app ) were highlighted in Table 2.
The higher value of binding constant and lower values of Stern-Volmer's quenching constant and apparent binding constant is evidence of the fact that IrL1 complex interacts with DNA via covalent bond and not via intercalation mode.
BSA binding study. Upon excitation at 295 nm, the emission intensity of BSA at l em ¼ 350 nm decreased gradually on increasing the complex concentration, which conrmed that the interaction between complex IrL1 with BSA had occurred, as observed in Fig. 8. The Stern-Volmer quenching constant of these complexes with BSA (K BSA ) was calculated using the Stern-Volmer equation and the corresponding Stern-Volmer plots (Fig. 8, inset). The binding affinity (K) of the complexes was calculated from Scatchard plot analysis (Fig. 8, inset).
The complexes showed strong binding propensity with BSA, which is required for the transport of protein-bound complexes in biological systems. K BSA for complex IrL1 was found to be 5 Â 10 4 M À1 the K value was 6.3 Â 10 6 M À1 . The value of bimolecular quenching constant (k q ) calculated from K SV and s 0 (1 Â 10 À8 s) was observed to be 5 Â 10 12 M À1 S À1 . These values are higher than the maximum possible value for dynamic quenching (2.0 Â 10 10 L mol À1 s À1 ), 29,30 suggesting the involvement of static quenching mechanism by the present iridium(III) complexes.   The non-linearity of the Stern-Volmer plot of IrL1 is due to the formation of a ground state complex between the Ir(III) complex and BSA.
Co-localization study. To identify the subcellular localization of the IrL1 complex, the cells were treated with the complex and stained with Hoechst and further explored using uorescence microscopy. The complex exhibited cytoplasmic localization at 3 mM concentration with a green uorescence emission (Fig. 9). Mitochondria are one of the key targets for several anticancer drugs. Consequently, further experimental validation involved mitochondrial membrane potential (MMP) analysis by JC-1 staining and detection of ROS generation by DCFDA staining.
Mitochondrial membrane dysfunction study. The IrL1 complex demonstrated mitochondrial depolarization of MDA-MB-468 cells in a dose dependent manner. The cytoplasmic localization of the IrL1 complex as evidenced by sub-cellular localization study impelled us to explore the effect of the complex on cytoplasmic mitochondria. Consequently, the alterations in mitochondrial membrane potential (MMP, DJ m ) and associated mitochondrial dysfunction was demonstrated by JC-1, a cationic carbocyanine dye which exhibits potentialdependent accumulation in mitochondria. The cells treated with the mitochondrial uncoupler CCCP (carbonyl cyanide mchlorophenylhydrazone), which mediates the dissipation of mitochondrial membrane potential, served as positive control for the detection of mitochondrial dysfunction.
The ow cytometric quantication of JC-1-stained cells revealed a normal mitochondrial function in control cells devoid of any treatment exhibiting $94% JC-1 aggregates (red+ green uorescing healthy mitochondria). Impaired mitochondrial function was observed in only 3.30% of control cells with JC-1 monomers indicating green uorescence (Fig. 10B). Unstained control represented in Fig. 10A has been used for gating purposes. Conversely, the positive control with CCCP treatment, displayed a high percentage of cells ($79%) with damaged mitochondria indicating the presence of green uorescing JC-1 monomers against $20% cells retaining healthy mitochondria with red + green uorescing JC-1 aggregates (Fig. 10C).
Treatment of the MDA-MB-468 cells with IrL1 has ensued a dose dependent increase in JC-1 monomer expressing/green uorescing cells indicating damaged mitochondria (Fig. 10D-F). IrL1 complex at 1 mM concentration resulted in damaged mitochondria in 27.86% cells (Fig. 10D). However, the higher concentrations of the IrL1 complex at 3 and 5 mM induced alterations of mitochondrial membrane potential and associated damage in high percentage $47.52% and 67.02% respectively in MDA-MB-468 breast cancer cells (Fig. 10E and F). The proportion of green uorescing JC-1 monomers induced by high concentrations of the complex at 3 and 5 mM thereby indicates alteration in the MMP and mitochondrial depolarization and associated mitochondrial dysfunction leading to a dose dependent possible cell death meditated by the IrL1 complex comparable to the CCCP treated positive control cells (Fig. 10C).
ROS generation studies. The IrL1 complex exhibited a dose dependent reactive oxygen species (ROS) generation in the MDA-MB-468 breast cancer cells. The MDA-MB-468 breast cancer cells exhibited ROS generation suggestive of cellular stress leading towards cell death at elevated ROS levels.
Our results indicated a dose dependent increase in ROS production in the MDA-MB-468 cells treated with IrL1 concentrations 1 mM and 3 mM. The extent of ROS production is indicated by the 2 0 ,7 0 -dichlorouorescein (DCF) formed by the deacetylation and subsequent oxidation of 2 0 ,7 0 -dichloro-uorescin diacetate (DCFDA) through cellular esterases and ROS. 1 mM concentration of the complex has resulted in 55.01% DCF positive cells which has increased up to 70.67% DCF positive cells at 3 mM concentrations. ROS production was absent in untreated control. However, in the positive control, treated with H 2 O 2 , ROS production was high which is $99% (Fig. 11).
Cell cycle analysis. The IrL1 complex exerted a G 0 /G 1 cell cycle arrest at the highest tested concentration of 5 mM. The   16%), followed by a G 2 /M phase with $33.18% cells, and 31.66% of G 0 /G 1 cells (Fig. 12). Conversely, the treatment of IrL1 complex resulted in a decrease of G 2 /M phase cells upto $18.37% and a decrease of S phase cells upto $28.32% with the highest concentration of the complex i.e., 5 mM. Remarkably, 5 mm concentration considerably increased G 0 / G 1 phase cells (53.32%), indicative of a substantial G 1 arrest of MDA-MB-468 breast cancer cells mediated by the IrL1 complex.
Annexin FITC/PI assay. The Annexin FITC/PI assay has demonstrated signicant apoptotic initiation of IrL1 treated MDA-MB-468 breast cancer cells. At 3 mM concentration of  the complex, $34.89% of cells were found to be displaying early apoptotic phase and $9.49% cells exhibited late apoptotic phase (Fig. 13). The viable cell population constituted $51.37% which supports the IC 50 concentration of 3.673 mM demonstrated by IrL1 on MDA-MB-468 cells thereby corroborating the IC 50 data at this particular concentration.

Materials and methods
In all the experiments, the reagents and solvents used were of the highest grade and best commercial quality. All organic solvents used throughout the chemical synthesis and chromatography procedures were of analytical grade and used without  further purication as received from E. Merck (India). Pentamethylcyclopentadienyl iridium(III) chloride dimer, 1,10phenanthroline-5,6-dione, a-naphthaldehyde, 9-anthraldehyde, chromone-3-carboxaldehyde, indole-3-carboxaldehyde and benzothiazole-2-carboxaldehyde were procured from Sigma Aldrich Chemical Ltd, Merck and Spectrochem. Thin layer chromatography was performed on pre-coated silica gel 60 F 254 aluminium sheets (E. Merck, Germany) and the solvent system was an ethyl-acetate-methanol mixture. 1 2 , was dissolved in about 10 ml of methanol in a 50 ml round bottomed ask and was stirred continuously for 5-10 min to dissolve the reactant. To the completely dissolved solution, 2.1 equivalents of the previously synthesized ligands (L1-L5) were added and stirred at room temperature. Aer 90 min, 2.5 equivalents of ammonium hexauorophosphate (NH 4 PF 6 ) (13.3 mg, 0.082 mmol) was added as ligand exchange salt in order to increase crystallinity and hence, purity of the product and again the reaction mixture was stirred for 90 min more at room temperature. The progress of reaction was conrmed by TLC. Aer complete conversion of the starting materials to the desired product, the solvent was evaporated under reduced pressure. The crude product was washed with hexane and further recrystallized from diethyl ether/methanol (1 : 1) solvent system. Finally, the complexes (IrL1-IrL5) were obtained as light brown crystals with high yield (90-92%).

Cytotoxicity studies
Cytotoxicity study of all the synthesized compounds against triple negative breast cancer cells MDA-MB-468 and immortalized human keratinocyte cell line HaCaT were done with a drug incubation period of 48 h by following standard procedure that were mentioned in ESI. † Preliminary studies were done to understand the mode of action of the complex to induce cytotoxicity using ow cytometric methods.

Mitochondrial membrane dysfunction assay
Alterations in mitochondrial membrane potential (MMP, DJ m ) and associated mitochondrial dysfunction was demonstrated by JC-1, a cationic carbocyanine dye. The cells were treated with the mitochondrial uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone), which mediates the dissipation of mitochondrial membrane potential, served as positive control for the detection of mitochondrial dysfunction. The ow cytometric quantication of JC-1 stained cells revealed a normal mitochondrial function in control cells and the results obtained from the quantied JC-1 stained cells among IrL1 treated cells were compared with the same of the control.

Imaging studies
Colocalization study and ROS generation studies were performed using live cell lines in their log phase.

Statistical analysis
As the study had more than one group, one way ANOVA was used for statistical analysis. The p value <0.05 was considered as signicant.

Conclusions
Five Ir(III)-imidazophenanthroline complexes were synthesized and characterized successfully. Our study has demonstrated the cytotoxic potential of IrL1 complex on MDA-MB-468 breast cancer cells in a dose dependent manner. The subcellular localization study of the complex revealed the localization of the compound in cytoplasm thereby pointing to a possible mitochondrial localization and consequent mitochondrial dysfunction. Subsequent analysis to demonstrate the alterations in MMP and ROS generation mediated by the IrL1 complex has revealed a signicant increase in mitochondrial dysfunction and a resultant increase in ROS production suggestive of the mitochondrial targeting potential of the IrL1 complex. The Annexin V-FITC/PI assay has validated the cytotoxic potential, along with the IC 50 dose of the complex on MDA-MB-468 breast cancer cells by initiating apoptotic pathway probably due to the cellular energetic stress triggered by elevated ROS levels. Furthermore, the IrL1 complex mediated a substantial G 1 phase cell-cycle arrest of MDA-MB-468 cells at the highest tested concentration of 5 mM. The study ndings support the prospective therapeutic potential of the IrL1 complex in the treatment and eradication of triple negative breast cancer cells.

Conflicts of interest
There are no conicts to declare.